The present invention describes novel devices and methods for detecting small labels using electronic sensors. Detecting the labels is of interest because they allow one to “see” or “quantify” tiny objects that would otherwise not be visible or detectable. For this to work, the labels must be “bound” to the object of interest. Or, sometimes, the labels are of interest in and of themselves. The object of interest could be a living cell or bacterium in the field of microbiology; a protein, nucleic acid, or antibody in the field of biochemistry; or almost any chemical species analyte.
In any case, the labels are very small particles. Here a particle is defined as a small physical object, typically with a longest dimension less than 1 mm. Many particles are roughly spherical, though there are also oblate spheroids, needles, cubes, and all manner of three dimensional shapes. The word “bead” is sometimes used to mean a particle.
A label is a specific kind of particle, one that gives off a measurable effect. It is this measurable effect that makes the label useful. In some cases, the measurable effect is light given off by a label dye. If enough label dye is present, the effect can be observed with the naked eye of a person looking at a jar of liquid. There are many cases, however, where the volume of fluid to be analyzed is very small, and the number of detectable objects is also very small, so the naked eye is not effective at detecting the object of interest. Furthermore, it is of great advantage to obtain data about the object of interest in the form of electronic digital data.
A step-by-step diagram is given in FIG. 1a to outline a process for using labels to generate electronic data.
Step 1. Energize the label.
Step 2. The label emits an effect.
Step 3. An electronic sensor receives the effect and converts it to an electronic signal.
Step 4. A detector circuit designed to work with the sensor converts the raw sensor signal to usable information. This data, then, can be used by a client (either another machine, or a person).
FIG. 1b shows, in simplified form, the physical components involved in performing the 4-step procedure 300 outlined in FIG. 1a. 
An electromagnetic effect field 314 is defined as: a distribution, in space and time, of electric and/or magnetic fields. A key feature of these effects is that they can be detected at a distance. Common examples of sources for such electromagnetic effect fields include: an electron has a stray electric field in its vicinity; a label with a net magnetic moment has stray magnetic fields in its vicinity; a photon is a massless label having energy that is observable as coupled oscillating electric and magnetic fields.
An electromagnetic effect field label 313 is defined as: a label that, under certain circumstances, gives off an electromagnetic effect field. These kinds of labels are very useful for analytical work because the detector does not have to be in direct contact with the label.
An electromagnetic effect field sensor 315 is defined as: an electronic device which, upon the impingement of an electromagnetic effect field, induces an electronic signal in a detector circuit. Examples of electronic sensors, and the electromagnetic effect fields for which they are designed to respond are include: magnetoresistance, magnetic field; hall effect, magnetic field; electrodes, electric field; Field effect transistor (FET), electric field; photodiode, photon; inductor, magnetic field; respectively.
An example of an electronic sensor that is NOT an electromagnetic effect field sensor is one where the labels themselves are part of the sensor or detector circuit. For example, DNA is known to have a net electrical charge, and electrodes may be used to measure this charge by applying an electric field between the electrodes and measuring the resulting electrical current flow.
An electromagnetic effect field detector circuit 317 is defined as: a circuit specifically designed to create electronic data based on the output of the electromagnetic effect field electronic sensor. This circuit typically has electrical connections for: power supply, ground, positive and negative inputs for one or more electromagnetic effect field sensors plus possibly a third terminal for voltage biasing, an amplifier stage, and an analog-to-digital (ADC) conversion stage. It may also have switching circuits that enable the collection of electronic signals induced by a plurality of sensors. The electronic data from the detector circuit is typically in digital form.
A control system 318 is used to collect data from the detector circuit, analyze it, and pass the result to a client (either a person or separate system). Electrical and/or radio linkages are provided by connections 316 and 319.
An electromagnetic effect field label excitation source 311 is defined as: a source of electromagnetic energy 312 that may be imparted upon an electromagnetic effect field label 313 in such a way to induce that label to give off its specific electromagnetic effect field, 314. For example, some magnetic labels give off an electromagnetic effect field in direct proportion to the magnitude of the ambient magnetic field present at the label. A magnetic field excitation source provides a controlled addition to the ambient field. Another example: a fluorescence effect label gives off photons of a certain energy after being irradiated with photons of a specific higher energy. A photonic excitation source provides a controlled addition of specific higher energy photons to the fluorescence effect label.
Many labels give off some value of their electromagnetic effect field in the absence of any excitation source. Consequently, excitation source 311 is not always needed in a detection instrument 310. For example, some magnetic labels are permanently magnetized. Some molecules exhibit “autofluorescence”.
Micro- and nano-magnetic labels are used commonly in biochemical assays as a means to capture, concentrate, and manipulate target analytes. They are also increasingly being used as detection labels for assay readout. These detection applications benefit from the large magnetic field signature generated by the labels when magnetized by an applied magnetic field, and by the very low incidence of background magnetic signals. Together, these features make magnetic detection attractive for a range of bioanalytical applications.
Several detection platforms can “see” and quantify magnetic labels, including magnetic microchips, SQUID magnetometers, scanning probes, and induction techniques. The focus of this disclosure is on the microchip-based devices. Magnetic microchip detectors are tiny, inherently rugged, and very low cost when produced in large quantities. They are made using manufacturing technology that is well developed. These sensors are incorporated into applications ranging from implantable medical devices, hearing aids, automobiles, and magnetic hard disk drives.
In-Vitro diagnostic applications present some exciting opportunities for magnetic microchip-based detection. Recent coil-based detector systems have generated highly quantitative results from lateral flow strip assays. The biochemical resolution of these assays is limited more by the lateral flow strip technology than the detection technology. Rapid improvement along several fronts (membrane properties and uniformity, magnetic label performance, micro-manufactured substrates, etc.) make it likely that taking full advantage of magnetic detector capability will be desirable in the near future. Magnetic microchips have the potential to build on these accomplishments by increasing the numerical precision and spatial resolution of the assay readout. By providing highly multiplexed reader element, many spots and lanes can be quantified on the same strip. Furthermore, the ultra-low cost of detector chips makes them candidates for incorporation with the assay strip as a consumable item such as a disposable assay cartridge.
The combined qualities of readers and detectors make magnetic microchips ideally suited for de-centralized use, and in disposable assay formats. Already popular examples of applications like this are the pregnancy test (visual detection) and glucose monitoring (electrochemical). Both of these are already well served by the existing technology. But their technology will not be easily adapted to multiplexed assays, and to assays needing precision. Disposable magnetic assay chips, then, are likely to find their best use for small panels (in the range of 2 to 100 analytes) of immunoassays on a single-device. Markets for this technology include tests for food and water safety, agriculture, homeland defense, allergy, as well as many biomedical and veterinary medical uses.
The basis for using magnetic labels for biological assays is that they can be attached to the analyte through a biochemically specific binding mechanism. Assuming this capability exists for a given analyte, the challenge is then to count the labels in a way that provides data that is meaningful for the assay result. In order to illustrate the magnetic detection mechanisms, a simple example will be presented: detecting a single magnetic label bound to a magnetoresistive sensor sandwiching a captured analyte. This single-label example is not practical for most assay applications, but it provides a framework for understanding more realistic situations such as detecting large number of immobilized labels, and moving labels. Then are given some examples of detection of 2 dimensional label layers (e.g. surface immobilization assays), and finally 3 dimensional plugs (e.g. lateral flow strips).
Single Magnetic Label Detection.
Magnetic labels become magnetized (magnetization is induced in their volume) in the presence of an applied magnetic field. The direction of the magnetization is parallel to the applied field. The magnitude of magnetization is linearly proportional to the applied field. Thus, the magnetization is zero when the applied field is zero. (Note: we limit this discussion to paramagnetic labels. Some labels are ferromagnetic and/or permanent magnets, though these are not very commonly used.) The magnetic fields in the vicinity of a magnetic label are the sum of the externally applied field Happlied, and the stray fields from the label Hlabel. The Happlied is usually from a magnetic field source that is far enough away to be considered spatially uniform in the vicinity of the label. But Hlabel varies strongly both in magnitude and direction. These fields and a magnetized label are shown above a sensor in FIG. 1c. Typical magnetic field sources are passing electrical current through a wire or coil of wire, using a permanent magnet, or both.
There are several microchip-based magnetic sensing technologies that could be used in biosensors including Hall effect, anisotropic magnetoresistance (AMR), Giant Magnetoresistance (GMR), and Magnetic Tunnel Junctions (MTJ). This disclosure focuses on GMR but is not meant to exclude the use of the others, which may have relative advantages in certain cases. A GMR detector is a multilayer metal film whose total thickness is in the range of 10 to 100 nm. Resistors are formed from the film using standard semiconductor wafer processing techniques such as photolithographic patterning and ion mill etching. Patterned resistors are about 1 micron wide, and arbitrarily long. The total resistance of a typical GMR resistor is about 10 ohms times the length:width ratio. For example, a 2 micron×100 micron resistor has a nominal resistance of (100 microns/2 microns)*10 ohms per square=500 ohms. By design, the GMR sensor is sensitive primarily along one axis in 3 dimensional space. This direction is usually in the plane of the film, but can be either parallel or perpendicular to the long dimension of the resistor. The electrical resistance of the GMR sensor is used in a circuit to infer the magnitude of the total magnetic field at the sensor location. A constant current is passed through the resistor, the voltage measured; the resistance is the voltage/current. This voltage is independent of the frequency of measurement, and exists at “DC.”
The Hlabel has the shape of a “dipole” field, much like the magnetic field of the Earth. Its magnitude drops off as the function (r/d)3, where d is the distance from the label and r is the label radius. Also, the field from the label is not uniform in the plane of the sensor. Clearly, the magnetic field effect of the label is greatest in the region immediately beneath it, and decreases rapidly as one moves out from underneath it. So the sensor resistance only changes appreciably in that region. Ignoring, for a moment, the non-uniformity of Hlabel, one can see the effect of the label on magnetic fields detected by the sensor. Referring to FIG. 2, one can see that for some Happlied, there is an Hlabel in the opposite direction that decreases the total field at the sensor. This effect is measured as a voltage change by electronic instrumentation.
If the label is much smaller than the total size of the sensor, the total fractional resistance change will be much smaller than if the label was the same size or larger than the sensor. In fact, the total resistance change it is roughly proportional to the fraction of the sensor area that the label covers. This is a very useful feature for measuring more than one label.
Label Layers.
In most bioanalytical applications, detecting a single label is not useful. Rather, it is necessary to measure, within the detection region, the surface density of labels in a quantitative way so that the concentration of some analyte can be inferred. A drawing of a sensor with more than one label above it is shown in FIG. 3. Notice that the sensor area can be extended almost arbitrarily by patterning it in a “serpentine” shape. Also, it is possible to create large arrays of very small sensors, which have both high precision and large area. Assay conditions must be set to match the analyte concentration range of interest, so that after the time interval of conjugation and labeling, the number of bound labels is somewhere between zero and full coverage. The total resistance change of the sensor then corresponds to the number of labels bound there.
Some early examples of this arrangement were demonstrated using GMR sensors under a 200 micron diameter circular spot to detect 2.8 Dynal labels bound to the sensor through a specific binding of captured DNA [Baselt U.S. Pat. No. 5,981,297]. The dynamic range of the detector was from 10 labels to about 5000 labels, nearly 3 orders of magnitude. This level of label detection corresponded to a maximum concentration of 100 fmolar DNA as measured using optical detection. An analogous result was obtained using ˜60 nm diameter labels in order to quantify the immunological interaction between surface-bound mouse IgG and α-mouse IgG coated on superparamagnetic labels.
Spots and Plugs.
Three-dimensional sample volumes are, of course, important in bioanalysis. It is possible to use GMR technology to measure the magnetic content of very small volume like those in a microfluidic channel or lateral flow membrane. Detecting a magnetic plug or volume works the same way as a single label, or layer of labels, in that the sensor detects changes in the local magnetic field due to the presence of magnetic material there. The complicating fact is that not all of the magnetic material can be said to be at the same distance from the sensor (recall: the stray magnetic field from a label goes as (r/d)3). The fact that the math is more complicated does not mean that the detector won't work; rather, that more care is required in sensor calibration and data analysis.
Magnetic Labels in Lateral Flow Strips.
A complete system for detecting magnetic labels in lateral flow strips has been developed and is being marketed by MagnaBioSciences. Descriptions of their system have been published [RT LaBorde and B O'Farrell, “Paramagnetic Particle Detection in Lateral-Flow Assays,” IVD Technology 8, no. 3 (2002): 36-41. B O'Farrell and J Bauer, “Developing highly sensitive, more reproducible lateral-flow assays Part 2: New challenges with new approaches”, IVD Technology 12, no. 6 (2006): 67. U.S. Pat. Nos. 6,046,585, 6,607,922]
This system has been used successfully for very quantitative work on e-coli according to the manufacturers, out-performing two reference systems for the application. The physics of detection is different for the MagnaBioSciences system than for the GMR system. The magnetic labels are paramagnetic, just as are discussed above. But the detectors are loops of wire, or inductors. The voltage induced in a loop of wire is proportional to its area, the frequency and magnitude of the oscillating magnetic applied field. There is no detected signal in a loop or coil if the field does not change in time. The bioanalytical volume being measured in this case is on the order of 1 mm×3 mm×0.4 mm. The first dimension is the width of the capture stripe on the strip; the second and third dimensions are the strip width and thickness, respectively. The pickup coils for this system must be well matched to the sample dimensions.
Most of the early demonstrations using GMR in bioanalytical applications assumed that the sample handling would be done in a lab-on-a-chip type device. Here, the passive wicking action of lateral flow strips is replaced by active pumping and/or vacuum. Well defined reservoirs and microfluidic channels replace the spots and strips. Consequently, labs-on-a-chip have the ability to perform a greater variety of functions, and do them much more precisely, but at the expense of greater product complexity.
Ferrofluid plugs of magnetic nanolabels (aqueous suspension of 10 nm magnetite labels, Ferrotec EMG 507) diluted to 1.2% v/v magnetite with a Tris-buffered fluorescein solution (3 mM after mixing, pH 8.0) for fluorescence imaging) were detected by a GMR sensor beneath a microfluidic flow channel. The plug dimensions were 13 microns×18 microns×85 microns for a total volume of 20±3 pL and contains about 5×108 10 nm labels. The first two dimensions are the channel width and depth, respectively, while the third is the plug length. The individual labels are many times further away from the sensor than their diameter. While this is not entirely analogous to the lateral flow strip example, it does show that the GMR sensor is capable of detecting and analyzing “composite plugs” nearby in contrast to counting single labels.
Microchip-Based Magnetic Sorting and Concentration.
Most magnetic labels are designed and used primarily for capturing and concentrating analytes. So it is natural to adapt macro-scale magnetic force systems to miniature bioanalytical systems. Electrical current passing through a wire generates magnetic fields that are directed around that wire. This means that wires on a circuit chip generate magnetic fields that impinge upon the magnetic label on the chip or nearby. When combined with other field sources, such onchip wires can be used to magnetize labels and attract or repel them. The magnetic forces are sufficient to enable the sorting of magnetic objects in flow streams [Tondra, et. al. “Design of Integrated Microfluidic Device for Sorting Magnetic Beads in Biological Assays,” IEEE Transactions on Magnetics, 37, no. 4 (2004): 2621-2623. N Pekas, et. al, “Magnetic particle diverter in an integrated microfluidic format;” Journal of Magnetism and Magnetic Materials, 293, (2005): 584. U.S. Pat. No. 6,736,978, Porter et al.; U.S. Pat. No. 6,875,621, Tondra]. These patents are incorporated herein by reference. Combining the ability to direct magnetic labels in flow along with a means to detect them downstream presents the opportunity to count magnetically labeled analytes as they flow by.
Cell sorting and detection has been demonstrated using this technique. Since single labels can be detected in flow, it is possible to make ultra-miniature flow detection systems for molecular analysis using magnetic label-carriers. For additional background, see the excellent review articles on magnetoresistive-based sensing [DL Graham, H A Ferreira and P P Freitas, “Magnetoresistive-based biosensors and biochips,” Trends in Biotechnology 22 (2004): 455-462] and microscale sorting [D L Graham, H A Ferreira and P P Freitas, “Magnetoresistive-based biosensors and biochips,” Trends in Biotechnology 22 (2004): 455-462].
Magnetic concentration in a flow strip device is described in U.S. Pat. No. 6,136,549, Feistel, but the detection means in that patent is optical, not magnetic. Details of flow permeable membranes as they are used with optical detection are described in U.S. Pat. No. 7,141,212, Catt, describes flow membranes, systems for optical reading. Electronic reading of fluorescently labeled assays is described in U.S. Pat. No. 5,837,546 by Allen.